Mohsen Najafi Najafi - Razi Vaccine and Serum Research Institute. Mashhad Branch, Agricultural Research, Education and Extension Organization (AREEO), Mashhad, Iran
Mohammad Hemmaty - Razi Vaccine and Serum Research Institute. Mashhad Branch, Agricultural Research, Education and Extension Organization (AREEO), Mashhad, Iran
Khadijeh Moridi - Department of Microbiology and Virology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran ۳ Antimicrobial Resistance Research Center, Buali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran ۴

BACKGROUND: Clostridium septicum has played a significant role as a causative agent of many acute fetal diseases in man and animals. Alpha- toxin is the main factor in the pathogenesis of C. septicum with hemolytic, necrotic and lethal activities. OBJECTIVES: The study was designed to evaluate alpha-toxin purification and antibody production rate against a local strain of C. septicum NH2 which could be applied in diagnosing kits, potency test of the vaccines, and other related applications. METHODS: Local strain of C. septicum NH2 was cultured in liver broth. Alpha-toxin in supernatant purified by three steps: the first step was done by 25% and 60% of ammoniums sulfate precipitation and continued by DEAE-Sephadex ion exchange chromatography, and finally finished in gel filtration on Sephadex G-50. Alpha-toxin was assayed in all steps and purification procedures were analyzed by SDS-PAGE. After immunization of rabbits with alpha- toxin and serum collection, immunoglobulin was separated by three purifying steps: ammoniums sulfate, ion exchange chromatography, and gel filtration. Serum purification process was evaluated by electrophoresis, double immunodiffusion (DID), single radial immunodiffusion (SRID), western blot, and SDS-PAGE. RESUTLS: SDS-PAGE results showed the alpha-toxin and anti-alpha-toxin were purified partially. Double immunodiffusion and single radial immunodiffusion methods detected the specific antibody. Heavy and light chains of anti-alpha-toxin separated by 2ME in electrophoresis reacted with 48 kDa alpha-toxin during the western blot without any reaction to other proteins in nitrocellulose paper. CONCLUSIONS: The present study showed a modified protocol for C. septicum alpha-toxin and anti-alpha-toxin production. The purification method is more economical and faster than previously reported procedures, and anti-alpha-toxin production is  an advantage in detection of C. septicum infection

alpha-toxin, anti-alpha-toxin, Clostridium septicum, polyclonal antibody, purification